Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity

Abstract

represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However, ribotoxins are larger, with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties. Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence. Examination of HtA Nterminal β-hairpin reveals differences in terms of length, charge, and spatial distribution.Consequently, four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [∆(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity, being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells. Keywords:Ribotoxins, hirsutellin A, Ribonucleases, rRNA, insecticidal. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 IntroductionRibotoxins are cytotoxic members of the family of fungal extracellular ribonucleases (RNases) best represented by RNase T1 (Yoshida 2001). They have been shown to be extremely toxic because they exert their ribonucleolytic activity just on a unique phosphodiester bond of the larger molecule of rRNA in the ribosome, leading to protein synthesis inhibition and cell death (Lacadena et al. 2007). This rRNA bond is unique because it is located at an evolutionarily conserved site, the sarcin-ricin loop (SRL), with essential roles in ribosome function (García-Ortega et al. 2010) and maturation (Lo et al. 2010), and it is also the target of the family of plant ribosome-inactivating proteins (RIPs), a group of glycosidases best represented by ricin (Nielsen and Boston 2001).In addition to their ribonucleolytic activity, fungal ribotoxins have the ability to cross lipid membranes in the absence of any known protein receptor, mainly due to their ability to interact with acid phospholipids Martínez-Ruiz et al. 2001;Oñaderra et al. 1993). This feature is the explanation of why these proteins display a remarkable but not highly specific antitumoral activity (Jennings et al. 1965;Lacadena et al. 2007;Olmo et al. 2001;Olson and Goerner 1965;Turnay et al. 1993).α-Sarcin is the most extensively characterized ribotoxin (Lacadena et al. 2007), but many others have been identified and/or characterized in different fungal spec...

Fungal Ribotoxins: A Review of Potential Biotechnological Applications

“…The equivalent residues in HtA (His 42, Glu 66 and His 113) were identified by structural comparison, showing that the catalytic residues are conserved, but also presenting other features closer to the T1-like RNases (Phe 126 instead of the equivalent Leu 145, for example) or even some others which were completely new (Asp 40 instead of Tyr 48) [ 31 ] ( Figure 1 B). Mutagenesis studies have indeed revealed that the active site of HtA would provide a more adaptable microenvironment than the one described for α-sarcin being able to better accommodate electrostatic and structural changes [ 31 , 32 , 33 ].…”

“…It is in this region where HtA and restrictocin show more variability when compared to α-sarcin. The N-terminal β-hairpin of HtA is much shorter ( Figure 1 ), but this truncation appears to be compensated by the extension of loop 5, which also exhibits a higher amount of positive charges [ 32 , 33 ].…”

“…Soon after its discovery, HtA was related to ribotoxins due to its ability to specifically cleave rRNA on Spodoptera frugiperda cells (Sf9), inhibiting cell growth [ 19 , 55 ]. HtA was then subjected to a fine structural and functional characterization, demonstrating that despite its smaller size and low sequence identity, it was a ribotoxin [ 5 , 31 , 32 , 33 , 61 ].…”